Study on α-dystroglycanopathy related enzymes in zebrafish

三重大学大学院生物資源学研究科博士後期課程生物圏生命科学専攻Muscular dystrophies are genetic diseases characterized by progressive muscledegeneration and muscular weakening. They can be classified into a number of diseasetypes, and some causative genes have been identified. Defects in glycosylation of -dystroglycan ( -DG), one of the dystrophin-glycoprotein complex (DGC)components, are responsible for certain congenital muscular dystrophies includingdiseases like Walker-Warburg syndrome (WWS) and muscle-eye-brain disease (MEB),so-called -dystroglycanopathies. -DG has unique glycans whose structure isSia 2-3Gal 1-4GlcNAc 1-2Man 1-Ser/Thr, and this glycan is required for binding tobasal lamina proteins such as laminin, perlecan and agrin. ProteinO-mannosyltransferases (POMT1 and POMT2) catalyze the first step in O-mannosylglycan synthesis on -DG only when they are co-expressed, and defects of POMT1 orPOMT2 result in WWS. O-Mannose -1,2-N-acetylglucosaminyltransferase 1(POMGnT1) catalyzes the transfer of GlcNAc from UDP-GlcNAc to the proteinO-mannosyl residue and it is responsible for MEB. Muscular structure duringembryogenesis has been well-studied in zebrafish that is an attractive model forinvestigation of genes involved in muscular development and degeneration.In this study, we cloned complete cDNAs encoding both zebrafish POMT1(zPOMT1) and zPOMT2, and zPOMGnT1 and investigated expression patterns of thosegenes in adult organs and tissues and during embryogenesis by quantitative RT-PCR andwhole-mount in situ hybridization. Each gene was expressed in all adult tissues,especially in ovary, and they were expressed ubiquitously throughout earlydevelopmental stages. Knockdown analysis using antisense morpholinooligonucleotides (MOs) was performed. As a result, while zPOMT2 morphant showed2more severe phenotypes with bended body, edematous pericardium and abnormalpigmentation of eyes, than zPOMT1 morphant with curved tail, edematous pericardiumand small eyes. Morphant of zPOMGnT1 had bended body, small eyes and edematouspericardium. Furthermore, we carried out immunostaining of tissue sections usinganti-glycosylated -DG antibody IIH6. IIH6 immunoreactivity was decreased in eachmorphant. We also examined whether they had enzymatic activity. zPOMGnT1expressed in human embryonic kidney 293 T (HEK293T) cells revealed enzymaticactivity, whereas zPOMT1 and zPOMT2 showed high levels of enzymatic activity onlywhen zPOMT1 and zPOMT2 were co-expressed in HEK293T cells. More interestingly,even though either human POMT1 (hPOMT1) or hPOMT2 was combined with eitherzPOMT1 or zPOMT2, there were still high enzymatic activities in HEK293T cells.Next, we considered that zebrafish is useful to produce transgenic fish byinjecting trace amount of plasmid DNA into fertilized eggs. Therefore, we investigatedwhether human POMGnT1 (hPOMGnT1) could be expressed functionally in zebrafishembryos. We detected the recombinant hPOMGnT1 in zebrafish embryos by means ofwhole-mount in situ immunostaining and Western blot analysis using anti-hPOMGnT1antibody. As a result, both analyses revealed that the embryos transiently expressing therecombinant hPOMGnT1 showed higher levels of POMGnT1 activity than the embryosinjected by a control plasmid. These results represented that O-mannosylation wasessential modification of proteins during early developmental stages in vertebrates andthat zebrafish was a fundamental resource for the study of vertebrate O-mannosylation.Finally, we investigated and analyzed the promoter regions of zPOMT1,zPOMT2, and zPOMGnT1, respectively. In comparison with these promoter regions, wefound several common transcription factor binding regions such as CdxA, GATA,3Nkx-2, and Oct-1. Meanwhile, MyoD-binding site was found in a position adjacent toE47-binding site in only zPOMGnT1 promoter and Tst-1-binding site was also found inonly zPOMT2 promoter. These results suggested that zPOMT1, zPOMT2, andzPOMGnT1 genes might be regulated by different manners and have several functionsother than protein O-mannosylation